Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses.

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

Purification of the structural and nonstructural proteins of St. Louis encephalitis virus.

We have developed a procedure for purifying both the structural and nonstructural proteins of flaviviruses from lysates of infected cell cultures. The procedure involves: immunoprecipitation to concentrate viral proteins and eliminate most of the cellular proteins, preparative polyacrylamide gel electrophoresis to separate the viral proteins, and hydroxyapatite chromatography, which eliminates most of the unlabeled cellular protein. This procedure offers an improvement over previous purification schemes in that there is no loss of viral proteins after the immunoprecipitation step, any combination of labeling isotopes may be used, and it is not necessary to soak proteins out of gel slices.

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.

A comparison of Saint Louis encephalitis and Sindbis virus RNA.

RNA isolated from purified St. Louis encephalitis and Sindbis virus particles was compared by cellulose CF-11 chromatography, and RNase T1 and pancreatic RNase A digestion. SLE RNA eluted from the cellulose CF-11 column as a molecule with very little secondary structure, while Sindbis appears to have some internal bonding. Ribonuclease digestion indicates that SLE RNA contains 2.4 per cent polyadenylic acid.

Group B arbovirus structural and nonstructural antigens. II. Purification of Saint Louis encephalitis virus intracellular antigens.

Three serologically distinct antigens were identified in homogenates of Saint Louis encephalitis (SLE) virus-infected cells after Brij-58 solubilization and diethylaminoethyl (DEAE)-cellulose chromatography. These antigens were designated as antigen I, II, and III. Immunodiffusion analyses showed that all antigen peaks which eluted from the DEAE-cellulose column contained the intracellular major envelope protein, antigen I. This protein was the only viral antigen which eluted at 0.125 M KCl in DEAE-cellulose column peak C. Optical density peak D, which eluted at 0.2 M KCl, contained both antigens I and III. Antigen III was purified from this column fraction approximately 200-fold by organic solvent extraction and chromatography on hydroxylapatite and Sepharose 6B. Purified antigen III had a molecular weight of 80,000 to 85,000 and contained only one antigenic determinant as judged by immunodiffusion analysis.

Saint Louis encephalitis viral ribonucleic acid replication complex.

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in the cytoplasm of infected cells associated with a membranous structure which sediments with an average value of 250S. The integrity of the complex is destroyed by detergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid (EDTA) which differentiates this structure from cellular polyribosomes. With cultures in which cellular RNA was highly labeled prior to infection, ribosomal RNA could not be demonstrated in the complex isolated from EDTA-sucrose gradients. Single-stranded 43S and the 26S and 20S forms of viral RNA were found in the complex. Viral RNA polymerase activity in sucrose-gradient fractions sedimented in the same region as the fractions which contained the pulse-labeled viral RNA. The polymerase incorporated (3)H-guanosine triphosphate into acid-precipitable material in the absence of added template. It was also found that the replication complex contains viral-specific proteins.